Limonoids from neem (Azadirachta indica A. Juss.) are potential anticancer drug candidates.

Neem (Azadirachta indica A. Juss.), a versatile evergreen tree recognized for its ethnopharmacological value, is a rich source of limonoids of the triterpenoid class, endowed with potent medicinal properties. Extracts of neem have been documented to display anticancer effects in diverse malignant cell lines as well as in preclinical animal models that has largely been attributed to the constituent limonoids. Of late, neem limonoids have become the cynosure of research attention as potential candidate agents for cancer prevention and therapy. Among the various limonoids found in neem, azadirachtin, epoxyazadiradione, gedunin, and nimbolide, have been extensively investigated for anticancer activity. Azadirachtin, a potent biodegradable pesticide, exhibits profound antiproliferative effects by preventing mitotic spindle formation and cell division. The antiproliferative activity of gedunin has been demonstrated to be mediated primarily via inhibition of heat shock protein90 and its client proteins. Epoxyazadiradione inhibits pro-inflammatory and kinase-driven signaling pathways to block tumorigenesis. Nimbolide, the most potent cytotoxic neem limonoid, inhibits the growth of cancer cells by regulating the phosphorylation of keystone kinases that drive oncogenic signaling besides modulating the epigenome. There is overwhelming evidence to indicate that neem limonoids exert anticancer effects by preventing the acquisition of hallmark traits of cancer, such as cell proliferation, apoptosis evasion, inflammation, invasion, angiogenesis, and drug resistance. Neem limonoids are value additions to the armamentarium of natural compounds that target aberrant oncogenic signaling to inhibit cancer development and progression.

Helicobacter pylori pathogen inhibits cellular responses to oncogenic stress and apoptosis.

Helicobacter pylori (H. pylori) is a common gastric pathogen that infects approximately half of the world's population. Infection with H. pylori can lead to diverse pathological conditions, including chronic gastritis, peptic ulcer disease, and cancer. The latter is the most severe consequence of H. pylori infection. According to epidemiological studies, gastric infection with H. pylori is the strongest known risk factor for non-cardia gastric cancer (GC), which remains one of the leading causes of cancer-related deaths worldwide. However, it still remains to be poorly understood how host-microbe interactions result in cancer development in the human stomach. Here we focus on the H. pylori bacterial factors that affect the host ubiquitin proteasome system. We investigated E3 ubiquitin ligases SIVA1 and ULF that regulate p14ARF (p19ARF in mice) tumor suppressor. ARF plays a key role in regulation of the oncogenic stress response and is frequently inhibited during GC progression. Expression of ARF, SIVA1 and ULF proteins were investigated in gastroids, H. pylori-infected mice and human gastric tissues. The role of the H. pylori type IV secretion system was assessed using various H. pylori isogenic mutants. Our studies demonstrated that H. pylori infection results in induction of ULF, decrease in SIVA1 protein levels, and subsequent ubiquitination and degradation of p14ARF tumor suppressor. Bacterial CagA protein was found to sequentially bind to SIVA1 and ULF proteins. This process is regulated by CagA protein phosphorylation at the EPIYA motifs. Downregulation of ARF protein leads to inhibition of cellular apoptosis and oncogenic stress response that may promote gastric carcinogenesis.

A Novel Probe for Spliceosomal Proteins that Induces Autophagy and Death of Melanoma Cells Reveals New Targets for Melanoma Drug Discovery.

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for approaches that identify new melanoma targets. We have previously reported a discovery of novel anti-melanoma compound 2155-14 (Onwuha-Ekpete et al., J Med Chem. 2014 Feb 27; 57(4):1599-608). In the report presented herein we aim to identify its target(s) and mechanism of action. METHODS: We utilized biotinylated analog of 2155-14 to pull down its targets from melanoma cells. Proteomics in combination with western blot were used to identify the targets. Mechanism of action of 2155-14 was determined using flow cytometry, RT-PCR, microscopy, western blot, and enzymatic activity assays. Where applicable, one-way analysis of variance (ANOVA) was used followed by Dunnett post hoc test. RESULTS: In the present study, we identified ATP-dependent RNA helicase DDX1 and heterogeneous nuclear ribonucleoproteins (hnRNPs) H1, H2 and A2/B1 as targets of anti-melanoma compound 215514. To the best of our knowledge, this is a first report suggesting that these proteins could be targeted for melanoma therapy. Mechanistic investigations showed that 2155-14 induces ER stress leading to potentiation of basal autophagy resulting in melanoma cell death in BRAF and NRAS mutated melanoma cells. CONCLUSION: Identification of mode of action of 2155-14 may provide insight into novel therapies against a broad range of melanoma subtypes. These studies were enabled by the novel probe derived from a mixture-based library, an important class of chemical biology tools for discovering novel targets.

Synthesis of 4-methyl-N'-(3-alkyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazides with antioxidant, antitumor and antimicrobial activities.

The structures of the newly synthesized 4-methyl-N'-(3-alkyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5a­5l) were confirmed by spectral and elemental analysis. The difference in the potency of activity against various free radicals, human cancer cells and microbial strains has been evaluated by SAR. Compounds with electron-donating methoxy (5i and 5c) and methyl (5h and 5b) substitutions at the para position of the phenyl showed excellent free radical scavenging effects. In the tested compounds, electron withdrawing fluoro (5k and 5e), chloro (5j and 5d), and bromo (5l and 5f) substitution at the para position of the phenyl ring attached to C-2 and C-6 carbons of the piperidine moiety outperformed cytotoxic and antimicrobial activities. Our findings suggest that the antioxidant, anti-tumor and anti-microbial activities of compounds 5a­5l create promising leads for the development of potent anti-tumor and anti-microbial agents.

Perspectives on Challenges in Cannabis Drug Delivery Systems: Where Are We?

Cannabis and its natural derivatives have emerged as promising therapeutics for multiple pathological and nonpathological medical conditions. For example, cannabinoids, the most popular and biologically active chemicals in cannabis, aid in many clinical ailments, including pain, inflammation, epilepsy, sleep disturbances or insomnia, multiple sclerosis, anorexia, schizophrenia, neurodegenerative diseases, anti-nausea, and most importantly, cancer. Despite the comprehensive benefits, certain aspects of cannabis present unique challenges in the medical cannabis landscape. Recent studies have highlighted the inherent challenges associated with cannabinoids' formulation like low solubility, rapid metabolism, poor bioavailability, and erratic pharmacokinetics - all of which contribute to the limited efficacy of cannabinoids. Several efforts are underway to address the bottlenecks and modify the formulations along with the delivery systems to achieve greater solubility/bioavailability, potency, and efficacy in treatment settings while minding the necessary standards for purity associated with the pharmaceutical industry. The current article presents a perspective on (1) a working knowledge of cannabinoids and their mechanisms of action, (2) the landscape of using medicinal cannabis for cancer-related medical conditions along with adversities, (3) current approaches, formulations, and challenges in medicinal cannabis delivery systems (oral, transdermal, pulmonary, and transmucosal), and lastly, (4) emerging approaches to improve delivery systems.

Nimbolide, a Neem Limonoid, Is a Promising Candidate for the Anticancer Drug Arsenal.

Nimbolide, a major limonoid constituent of Azadirachta indica, commonly known as neem, has attracted increasing research attention owing to its wide spectrum of pharmacological properties, predominantly anticancer activity. Nimbolide is reported to exert potent antiproliferative effects on a myriad cancer cell lines and chemotherapeutic efficacy in preclinical animal tumor models. The potentiality of nimbolide to circumvent multidrug resistance and aid in targeted protein degradation broaden its utility in enhancing therapeutic modalities and outcome. Accumulating evidence indicates that nimbolide prevents the acquisition of cancer hallmarks such as sustained proliferation, apoptosis evasion, invasion, angiogenesis, metastasis, and inflammation by modulating kinase-driven oncogenic signaling networks. Nimbolide has been demonstrated to abrogate aberrant activation of cellular signaling by influencing the subcellular localization of transcription factors and phosphorylation of kinases in addition to influencing the epigenome. Nimbolide, with its ever-expanding repertoire of molecular targets, is a valuable addition to the anticancer drug arsenal.

Role of Bacterial and Viral Pathogens in Gastric Carcinogenesis.

Gastric cancer (GC) is one of the deadliest malignancies worldwide. In contrast to many other tumor types, gastric carcinogenesis is tightly linked to infectious events. Infections with Helicobacter pylori (H. pylori) bacterium and Epstein-Barr virus (EBV) are the two most investigated risk factors for GC. These pathogens infect more than half of the world's population. Fortunately, only a small fraction of infected individuals develops GC, suggesting high complexity of tumorigenic processes in the human stomach. Recent studies suggest that the multifaceted interplay between microbial, environmental, and host genetic factors underlies gastric tumorigenesis. Many aspects of these interactions still remain unclear. In this review, we update on recent discoveries, focusing on the roles of various gastric pathogens and gastric microbiome in tumorigenesis.

Bacterial CagA protein compromises tumor suppressor mechanisms in gastric epithelial cells.

Approximately half of the world's population is infected with the stomach pathogen Helicobacter pylori. Infection with H. pylori is the main risk factor for distal gastric cancer. Bacterial virulence factors, such as the oncoprotein CagA, augment cancer risk. Yet despite high infection rates, only a fraction of H. pylori-infected individuals develop gastric cancer. This raises the question of defining the specific host and bacterial factors responsible for gastric tumorigenesis. To investigate the tumorigenic determinants, we analyzed gastric tissues from human subjects and animals infected with H. pylori bacteria harboring different CagA status. For laboratory studies, well-defined H. pylori strain B128 and its cancerogenic derivative strain 7.13, as well as various bacterial isogenic mutants were employed. We found that H. pylori compromises key tumor suppressor mechanisms: the host stress and apoptotic responses. Our studies showed that CagA induces phosphorylation of XIAP E3 ubiquitin ligase, which enhances ubiquitination and proteasomal degradation of the host proapoptotic factor Siva1. This process is mediated by the PI3K/Akt pathway. Inhibition of Siva1 by H. pylori increases survival of human cells with damaged DNA. It occurs in a strain-specific manner and is associated with the ability to induce gastric tumor.

Helicobacter pylori pathogen regulates p14ARF tumor suppressor and autophagy in gastric epithelial cells.

Infection with Helicobacter pylori is one of the strongest risk factors for development of gastric cancer. Although these bacteria infect approximately half of the world's population, only a small fraction of infected individuals develops gastric malignancies. Interactions between host and bacterial virulence factors are complex and interrelated, making it difficult to elucidate specific processes associated with H. pylori-induced tumorigenesis. In this study, we found that H. pylori inhibits p14ARF tumor suppressor by inducing its degradation. This effect was found to be strain-specific. Downregulation of p14ARF induced by H. pylori leads to inhibition of autophagy in a p53-independent manner in infected cells. We identified TRIP12 protein as E3 ubiquitin ligase that is upregulated by H. pylori, inducing ubiquitination and subsequent degradation of p14ARF protein. Using isogenic H. pylori mutants, we found that induction of TRIP12 is mediated by bacterial virulence factor CagA. Increased expression of TRIP12 protein was found in infected gastric epithelial cells in vitro and human gastric mucosa of H. pylori-infected individuals. In conclusion, our data demonstrate a new mechanism of ARF inhibition that may affect host-bacteria interactions and facilitate tumorigenic transformation in the stomach.

Recruitment of DNA polymerase eta by FANCD2 in the early response to DNA damage.

How Fanconi anemia (FA) protein D2 (FANCD2) performs DNA damage repair remains largely elusive. We report here that translesion synthesis DNA polymerase (pol) eta is a novel mediator of FANCD2 function. We found that wild type (wt) FANCD2, not K561R (mt) FANCD2, can interact with pol eta. Upon DNA damage, the interaction of pol eta with FANCD2 occurs earlier than that with PCNA, which is in concert with our finding that FANCD2 monoubiquitination peaks at an earlier time point than that of PCNA monoubiquitination. FANCD2-null FA patient cells (PD20) carrying histone H2B-fused pol eta and wtFANCD2, respectively, show a similar tendency of low Mitomycin C (MMC) sensitivity, while cells transfected with empty vector control or pol eta alone demonstrate a similar high level of MMC sensitivity. It therefore appears that FANCD2 monoubiquitination plays a similar anchor role as histone to bind DNA in regulating pol eta. Collectively, our study indicates that, in the early phase of DNA damage response, FANCD2 plays crucial roles in recruiting pol eta to the sites of DNA damage for repair.